2.6. Characterization of the immobilized GluDH

The relative activities of free and immobilized His‐tagged GluDH were measured at the temperature range from 25 to 60℃, pH range from 4.0 to 10.6, respectively. The buffers used were Citrate buffer (pH 4.0–6.0), Sodium phosphate (pH 6.0 and 7.0), Tris‐HCl (pH 7.0–9.0), and Glycine‐NaOH (pH 9.0–10.6). Furthermore, the effect of different metal ions and surfactants on the activities of free and immobilized His‐tagged GluDH was examined. The concentration of Ca²⁺, Zn²⁺, Mn²⁺, Co²⁺, Mg²⁺, Li⁺, and EDTA were 5 mM, and that of the Tween 80 and Triton X‐100 were 1% (w/w), respectively.

The pH‐stability of the free and NF‐MNPs immobilized His‐tagged GluDH was tested by comparing the initial enzymatic activity and residual activity of free and immobilized enzyme after 24 h incubation in verifying pH buffer range from 4.0 to 10.0. Thermostability of free and immobilized His‐tagged GluDH was investigated by incubating them in 50 mM Tris‐HCl buffer (pH 8.0) at a specified temperature between 25 and 50℃. The reusability of immobilized His‐tagged GluDH was evaluated by repeated utilization of the immobilized His‐tagged GluDH to catalyze d‐glucose to gluconolactone at 45℃ and pH 8.0. The activity obtained in each round was compared with the initial activity to calculate the relative activity.

To study the binding specificity between the NF‐MNPs and His‐tagged GluDH, the commercial Ni‐NTA resin was also employed as a support to purify the His‐tagged GluDH from crude cell lysis. Specifically, about 20 mg NF‐MNPs were added in the 5 mL Eppendorf tube containing 2 mL cell lysis solution, and the mixture was incubated at 30℃ for 2 h. For contrast, 0.1 g wet weight commercial Ni‐NTA resin was mixed with 2 mL cell lysis and incubated at 4℃ for 2 h. Then, the immobilized GluDH were separated with the assistance of a magnetic field and washed twice with Tris‐HCl buffer. Subsequently, the GluDH was eluted with 0.5 mL elution buffer containing varied concentrations of imidazole from 20 to 500 mM in turn. Finally, the collected samples were analyzed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) to verify the binding specificity between the NF‐MNPs and His‐tagged GluDH.

The kinetic parameters for free‐state and immobilized GluDH were determined by measuring the activity in different substrate concentrations in the range of 0–40 mM. The V _max and K_m values were calculated by nonlinear regression with the fitting of the Michaelis–Menten equation.